Bacterial meningitis is a neurologically devastating and often life threatening illness. Rapid and accurate diagnosis is therefore of utmost importance in providing timely and optimal care. In contrast, viral meningitis, which often presents with the same clinical signs and symptoms as bacterial meningitis, is a self-limiting illness that does not require antimicrobial treatment and hospitalization. See, Cherry, J. D., Textbook of Pediatric Infectious Diseases, 3rd ed. Feigin, R. D., et al. (Eds.) W.B. Saunders Co., Philadelphia, pp. 439-445 (1992), the disclosure of which is incorporated herein by reference.
Currently, physicians in the emergency room setting are repeatedly faced with the diagnostic dilemma of how to clinically distinguish between bacterial and viral meningitis. This feat is virtually impossible with any degree of certainty. Therefore, a patient is often hospitalized and treated with intravenous antibiotics until the results of bacterial cultures from the cerebrospinal fluid ("CSF") are determined. This process generally takes between 48 to 72 hours to complete. The waiting period can therefore result in a tremendous increase in health care costs and the provision of suboptimal care, particularly since greater than 90% of meningitis cases are of a viral etiology.
The use of microscopy in the diagnosis of bacterial meningitis lacks both specificity and sensitivity and further requiring a high titer of bacteria in the CSF. An additional complicating factor is that prior treatment of the patient with antibiotics can lead to a false-negative result of both gram-stain and culture from CSF. For these reasons, physicians are hesitant to rely on culture results and will opt to complete a full 10-14 day course of intravenous antibiotics which in the majority of cases is not necessary.
The potential benefits of the polymerase chain reaction ("PCR") technique to provide identification of a specific bacterial or viral etiology affects all patients by making this distinguishing information available in a relatively short period of time. A viable PCR-based assay has the potential to influence the clinician's decisions of how to institute treatment while the patient is still in the emergency room. Since a PCR-based method of detection does not depend on the presence of viable organisms but instead relies on genetic material, a PCR-based technique is applicable in all patient cases, even when antibiotics were administered prior to CSF specimen collection. Some difficulties, however, are associated with PCR-based methods, such as false-positive results due to contaminating nucleic acids and inhibition of the PCR reaction due to complex samples as detailed below.
PCR-based assays for bacterial and viral pathogens that cause meningitis have been developed in recent years. Radstrom, P., et al., J. Clin, Microbiol. 32: 2738 (1994) the disclosure of which is incorporated herein by reference, describes a PCR strategy for the simultaneous detection in CSF of Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus agalactiae, and eubacteria in general. Their assay showed a high sensitivity of 0.94 and specificity of 0.96 when tested on 304 clinical CSF samples. Their approach included the use of a nested PCR strategy to detect the various pathogens. This technique, however, increases the risk of contamination of the PCR reactants with amplicons because the reaction tubes have to be opened between PCR steps when using such a strategy. Similarly, Hall L. M. C., et al., Eur. J. Clin. Microbial. Infect. Dis. 14: 1090 (1995), the disclosure of which is incorporated herein by reference, describe a similar PCR-based assay for the detection of bacterial pathogens that cause meningitis. Once again, a nested PCR technique was used with its intrinsic shortcomings.
Enteroviruses currently account for 80%-92% of all cases of aseptic meningitis for which an etiologic agent is identified as reported by Rotbart, H. A., et al., Clin. Inf. Dis. 20: 971 (1995), the disclosure of which is incorporated herein by reference. Several reverse transcriptase ("RT")-PCR-based assays for the diagnosis for enteroviruses have been developed in recent years. See, for example, Abzug, M. J., et al., J. Pediatr. 126: 447 (1995); Chapman, N. M., et al., J. Clin. Microbiol. 28: 843 (1990); Rotbart, H. A., J. Clin. Microbiol. 28: 438 (1990); and Zoll, G. J., et al., J. Clin. Microbiol. 30: (1992), the disclosures of which are incorporated herein by reference. Lina, B., et al., J. Clin. Microbiol. 34: 302 (1996), the disclosure of which is incorporated herein by reference, report the use of a PCR-based assay to detect enteroviruses from cerebrospinal fluid. In that assay different concentrations of enteroviruses were artificially inoculated into sterile CSF and the CSF was analyzed using RT-PCR in 13 different laboratories. The RT-PCR assay was found to be more sensitive than cell culture for enteroviruses. Yerly, S., et al., J. Clin. Microbiol. 34: 199 (1996), the disclosure of which is incorporated herein by reference, compared the RT-PCR-based assay to viral cultures in 38 patients with aseptic meningitis. The rates of enterovirus detection were 66% by PCR and 34% by culture.
There remains a need, however, for a PCR-based assay that can simultaneously detect and discriminate between the pathogens that cause bacterial and viral meningitis which in addition to being rapid, is not prone to contamination and which has increased sensitivity and specificity over other methods.
The present invention provides a PCR-based multiplex assay for bacterial and viral meningitis which allows for rapid discriminatory diagnosis between the viral and bacterial types of meningitis and which can provide the foundation data for a diagnosis even after the institution of antimicrobial therapy.